Total internal reflection microscopy is an observation method that allows nanoscale local excitation and has a high S/N ratio. The prime characteristics of this method are utilization of evanescent waves caused by total internal reflection of light at a boundary between two materials different in refractive index. When light is incident onto a boundary between a material 1 of refractive index n1 and a material 2 of refractive index n2 from the material 2 of the higher refractive index at a critical angle or more, the light is totally internally reflected at the boundary; this generates evanescent waves, which exponentially decays in the material 1 of the lower refractive index. The evanescent wave is light slightly leaking into a region of an extent of several tens to hundreds of nanometers from the total internal reflection boundary. Accordingly, total internal reflection microscopy generates evanescent waves at the boundary between a fluorescently stained sample and a slide glass, thereby allowing fluorescent observation with a high S/N ratio that is restricted to a significantly small part of the sample adjacent to the slide. This can be applied to single molecule observation.
As to applications utilizing the total internal reflection microscope, Non Patent Literature 1 describes observation of plasma membrane activity and single-molecule events in cell biology fields. Non Patent Literature 2 describes electrical characteristics of colloidal particles in electrochemical fields. Non Patent Literature 3 describes empirical elucidation of Brownian movement. The total internal reflection microscopes thus contribute to many fields. In recent years, application to nucleic acid sequence analysis (DNA sequencing) has been attempted. This will hereinafter be described.
Capillary sequencing, which is a combination of DNA fragment preparation mainly referred to as Sanger method and electrophoresis, is adopted as a present DNA sequencing method. Capillary sequencing has been utilized for human genome analysis and the like, and has yielded great results. However, in consideration of personal genome analysis from viewpoints of tailor-made medical care and the like, a technique has strongly been demanded that allows quick, simple and inexpensive analysis of DNA fragments much longer than those capable of being analyzed by one time of capillary sequencing. Conventional human genome analyses require approximately ten million dollars for analyzing genomes of one person. It is expected that realization of human genome analysis for a thousand dollars, which is a ten-thousandth thereof, dramatically advances applications of sequencing to medical fields. These demands cannot be met only with improvements of the conventional capillary method. Ultimately, if nucleic acids to be analyzed can be sequenced at a single molecule level without nucleic acid amplification, such as PCR, reagent fees become inexpensive because nucleic acid amplification is omitted, and this allows quick and simple sequencing. Further, single molecule sequencing negates the difference in amplification efficiency owing to nucleic acid amplification, thereby allowing highly precise quantification of the number of mRNAs appearing in cells in comparison with the conventional methods. Accordingly, single molecule DNA sequencing based on a novel system has been awaited.
As methods based on novel systems in order to realize this, a method of directly sequencing DNA using a scanning electron microscope, and a nanopore method of sequencing using a fact that voltage values of single strand DNA passing through a nanometer-sized pore are different among nucleotides A, G, C and T have been proposed. However, these methods include many technical problems. Accordingly, it is considered that realization of these methods is difficult.
As promising methods of DNA sequencing replacing these methods, massively parallel analysis methods using an optical technique have been proposed. Apparatuses based on principles of chemiluminescence or fluorescence have already appeared on the market from several companies. Characteristics of these methods are that segmentation of reaction fields using micro beads and micromachining techniques allows massively parallel analysis. Conventional capillary sequencing has improved analysis efficiency by means of multi-channels (to 384). However, the present system also allows massively parallel analysis of hundreds millions units or more, which is much more than the case of capillary sequencing. Accordingly, the readable length of nucleotides is 100 nucleotides or less, which is inferior to capillary sequencing capable of reading almost 1000 nucleotides. However, the throughput is, for instance, 100 nucleotides×hundreds million units (108), or 10 giga-(1010) nucleotides per day. Throughput one thousand times that of the capillary method can be expected. Further, the massively parallel analysis reduces the amount of reagent per sample, resultantly reducing the reagent cost. Accordingly, the analysis cost is approximately 100 thousand dollars per human genome of a person, which is approximately one hundredth that of capillary sequencing. However, since these methods amplify nucleic acids to be read and sequence the amplified nucleic acids, it is difficult to further reduce the analysis cost.
In order to attain further reduction in analysis cost, Non Patent Literature 4 has proposed a method of single molecule DNA sequencing according to a massively parallel analysis method using an optical technique. This method will hereinafter be described in detail.
Lasers of wavelengths of 532 and 635 nm are employed and used for detecting fluorescence of phosphors Cy3 and Cy5, respectively. A sample solution is sandwiched between two slide glasses, and then a single target DNA molecule is immobilized on a refractive index boundary surface between the slide glass and the sample solution on the solution side using biotin-avidin binding. Next, primers labeled with Cy3 are introduced in the solution by solution exchange so as to be in a certain concentration, which hybridizes a single Cy3-labeled primer molecule with a target DNA molecule to form a nucleic acid double strand. Subsequently, unreacted Cy3-labeled primer molecule is removed by a cleaning process.
The Cy3-labeled primer molecule hybridized with the target DNA molecule residing at a certain position in the evanescent field. Accordingly, the binding position of the target DNA molecule can be confirmed by detecting fluorescent. In a case where a plurality of Cy3-labeled primer molecules hybridized with the target DNA molecule exist in one scope of the evanescent waves, the entire positions of the Cy3-labeled primer molecules are grasped, thereby allowing subsequent sequencing to be performed in parallel. Further, in a case where a plurality of Cy3-labeled primer molecules hybridized with the target DNA molecule exist in one scope of the evanescent waves and across scopes, the entire positions of the Cy3-labeled primer molecules are grasped while the scope is moved by sliding a stage holding the slide glass, thereby allowing subsequent sequencing to be performed in a massively parallel manner. It is preferable to set the microscope at low power to widen the scope, in order to improve the throughput of sequence analysis. It is also preferable to increase the stage moving speed and to reduce moving time between the scopes incapable of fluorescent observation.
After verification of the positions of all the primer molecules, Cy3 is irradiated with high power excitation light for a certain time to fade fluorescence (quench fluorescence), thereby suppressing subsequent emission of fluorescence. The object of this is to prevent Cy3 in the previous process from being detected when Cy3 is used in and after the following process. In a case where a fluorochrome different from Cy3 is used in and after the following process, the fluorochrome is not necessarily to be quenched. However, there is a possibility that a fluorescent wavelength region of Cy3 overlaps with that of the other fluorochrome. Accordingly, it is preferable to quench the fluorochrome as much as possible.
Next, a solution including an enzyme for adding nucleotides to double-stranded nucleic acids, Cy5 fluorescence-labeled dNTPs (N is one type of A (adenine), C (cytosine), G (guanine) and T (thymine)) is introduced to be in a certain concentration by means of solution exchange. Only in a case of a complementary strand (A and T; C and G) with respect to the target DNA molecule, the fluorescence-labeled Cy5-dNTP molecule is taken into an elongating strand of primer molecules, which is one strand of the double strand nucleic acids. Typically, when the Cy5 fluorescence-labeled dNTP is taken into the elongating strand of the primer molecules, the enzyme tries to take in the next nucleotide. However, a mechanism is adopted in which a certain molecule is preliminarily bound to the position for the nucleotide of the Cy5-dNTP molecule, thereby preventing two nucleotides and more from being consecutively taken in. Subsequently, unreacted Cy5-dNTP molecules are removed by a cleaning operation.
Cy5-dNTPs taken into the elongating strand reside at specific positions in the evanescent field. This allows the binding position of Cy5-dNTP to be verified by detecting fluorescence. Further, identification of a position at which the binding position of Cy5-dNTP and the binding position of the target DNA molecule match with each other allows the sequence of the target DNA molecules immobilized at the prescribed positions in the evanescent field to be read. In a case where a plurality of Cy5-dNTPs taken into the elongating strand of the primer molecules exist in one scope of the evanescent waves, grasping of the positions of all the bound Cy5-dNTPs enables the sequence of the target DNA molecules to be read in parallel. In a case where a plurality of Cy5-dNTPs taken in the elongating strand of the primer molecules exist in one scope of the evanescent waves and across the scopes, grasping of the positions of all the Cy5-dNTPs by moving the stage holding the slide glass to move the scope enables the sequence of the target DNA molecule to be read in a massively parallel manner. It is preferable to set the microscope at low power to widen the scope, in order to improve the throughput of the sequence analysis. It is also preferable to increase the stage moving speed and to reduce moving time between the scopes incapable of fluorescent observation.
After verification of the entire sequence of Cy5-dNTP (one nucleotide), Cy5 is irradiated with high power excitation light for a certain time to fade fluorescence (quench fluorescence), thereby suppressing subsequent emission of fluorescence. In a case where a fluorochrome different from Cy5 is used in and after the following process, the fluorochrome is not necessarily to be quenched. However, there is a possibility that a fluorescent wavelength region of Cy5 overlaps with that of the other fluorochrome. Accordingly, it is preferable to quench the fluorochrome as much as possible. After quenching of Cy5, in order not to consecutively take in two nucleotides or more, the specific molecule bound to the Cy5-dNTP molecule is removed using means such as a catalyst or optical dissociation. This allows the next nucleotide to be elongated.
The above elongation reaction process of Cy5-dNTP is repeated sequentially on four types of nucleotides, for instance such as, dATP→dCTP→dGTP→dTTP→dATP, thereby allowing the nucleotide sequence of the immobilized target DNA molecule to be determined. The elongation reaction process of dNTP enables target DNA molecules to be sequenced in a massive parallel manner. The principle of single molecule sequencing has been described with the example of the fluorochromes of two colors, Cy3 and Cy5. However, fluorochromes are not limited to these two fluorochromes. The technique can be realized by another fluorochrome or a method. For instance, dNTPs are labeled with respective four types of different fluorochromes, negating the need to repeat elongation reaction of the aforementioned four types of nucleotides, dATP→dCTP→dGTP→dTTP→dATP. Accordingly, the throughput becomes four times faster according to a simple calculation. The primer molecules and dNTPs can be labeled with an identical fluorochrome (monochrome).
Non Patent Literature 5 has reported real-time single molecule sequencing as a method of single molecule DNA sequencing with a throughput higher than that of the above literature. Many conventional DNA sequencings utilize a DNA polymerase as an enzyme. However, in the method as with the above literature that performs elongation reaction and sequencing on each nucleotide, the ability that is included in the enzyme and consecutively takes in nucleotides is wasted. The ability of a single molecule of DNA polymerase to take in nucleotides is approximately 1000 nucleotides per second, which is capable of reading over 100 thousand nucleotides and further exerts significantly high fidelity. Thus, two techniques are adopted to consecutively elongate nucleic acid and perform real-time sequencing.
A first technique attaches a phospholinked nucleotide to a distal phosphoric acid, instead of attaching fluorescent labels to nucleotides, and separates the fluorochrome in a process that the enzyme takes in nucleotides. After the nucleotides have thus been taken in, completely natural double strand DNA remains. Fluorescence corresponding to the nucleotide when the enzyme takes in the nucleotide is detected in real-time, thereby allowing consecutive sequencing. Note that it is required to label the four types of nucleotides with different fluorescent labels. Only for a certain time until the enzyme takes in the fluorochrome, the fluorochrome exists at a specific position in the evanescent field. Accordingly, grasping the position at this time allows sequencing. After the fluorochromes are separated according to a second technique, the fluorochromes are adrift in the solution according to the Brownian movement. Accordingly, the fluorochromes do not affect sequencing. In contrast to the method of performing elongation reaction and sequencing on each nucleotide, this method negates the need of the process of quenching the fluorochrome by irradiation with high-power laser.
The second technique is a zero-mode waveguide technique allowing single molecule detection. This technique allows measurement of only a fluorochrome in a nanometer-sized pore. Accordingly, this allows measurement without removing, by a cleaning operation, the fluorochromes separated from the nucleotides and unreacted fluorescently labeled nucleotides not contributing to elongation reaction. These techniques suggest realization of a real-time DNA sequencing.
In a real-time single molecule sequencing, the elongation reaction proceeds consecutively. Accordingly, it is required to fix a normal scope until one sequencing is finished. Thus, in order to improve the throughput, it is effective to set the microscope at low power to widen the scope as much as possible. However, in Non Patent Literature 5, since an objective-type total internal reflection microscope is employed, it is limited to high magnification detection of 60× or more. Two types of total internal reflection microscopes used for single molecule sequencing and the like will hereinafter be described.
A presently used typical total internal reflection microscope is an objective-type total internal reflection microscope. The microscope adopts an inverted arrangement. The objective is positioned below a slide glass via immersion oil. Laser light for generating evanescent waves is obliquely incident from below the slide glass via the objective, thereby generating evanescent waves around a boundary on the slide glass where a sample is disposed. Because a space above the objective can freely be used, this arrangement has characteristics that are excellent in operability and convenience and further capable of acquiring a significantly bright fluorescent image. However, owing to limitation of the principle employing an oil-immersion objective with a high numerical aperture, there is a drawback of limitation to observation with a high magnification of 60× or more.
As another type of total internal reflection microscope without limitation to high magnification observation, a prism type total internal reflection microscope in which a laser is incident via a prism is used. In this microscope, a sample is sandwiched between two slide glasses or a slide glass and a cover glass; the prism is mounted on the upper slide glass; laser light for generating evanescent waves is obliquely incident from above the upper slide glass via the prism; this generates evanescent waves around a boundary of the slide glass contacting with the sample. This arrangement enables the laser light to be efficiently incident, thereby allowing observation with an S/N ratio higher than that of the objective type. Further, in contrast to the objective-type total internal reflection microscope, there is no limitation of magnification. Accordingly, low magnitude observation is also easy. The low magnitude observation widens a scope, thereby improving the throughput, for instance, in Non Patent Literature 5. Therefore, it can be said that the prism type total internal reflection microscope is more suitable than the objective type in view of improvement in throughput. However, in the prism type total internal reflection microscope, a space above the objective is occupied by the prism. Thus, there are drawbacks that operability of the sample and flexibility in arrangement of a specimen are significantly low. It is expected to develop a prism type total internal reflection microscope system that is excellent in operability of a sample and flexibility in arrangement of a specimen, easy to be used together with another optical observation method, and allows low magnitude observation.
Following efforts have been taken in order to improve operability of a sample and flexibility of arrangement of a specimen using a prism type total internal reflection microscope.
First, Non Patent Literature 6 has proposed a system in which an incident prism and an emission prism are cemented onto the undersurface of a slide glass, laser light is introduced into the slide glass from the incident prism to be totally internally reflected in the slide glass in a multiplexed manner, evanescent waves are generated around the upper surface of the slide glass to excite the sample during the multiplexed total internal reflection, the laser light wave-guided by the multiplexed total internal reflection is derived to the outside via the emission prism.
Non Patent Literature 7 has proposed a system in which an end of a slide glass is processed to form an inclined end surface, laser light is introduced into the slide glass from the inclined end surface and totally internally reflected in a multiplexed manner, evanescent waves are generated around the upper surface of the slide glass to excite the sample during the multiplexed total internal reflection, the laser light wave-guided by the multiplexed total internal reflection is derived from an end surface opposite to the inclined end surface to the outside.
These systems have characteristics that the space above the specimen is unoccupied and low magnitude observation is easy. However, since the thickness of the slide glass is limited to approximately 0.2 mm and thin, the number of multiplexed total internal reflections is increased. Accordingly, this tends to generate scattering light owing to the total internal reflections, attenuate wave-guided light, fade fluorescence of a sample, and reduce the S/N ratio. Further, since the incident and emission positions of laser light are fixed, it is required to move the objective in order to change the position of observing a sample and thus the operation is not easy. Accordingly, in a case of a prism type total internal reflection microscope, since a space above the objective is occupied by the prism and the space therebelow is occupied by the objective, operability of a sample and flexibility in arrangement of a specimen are low.